MAGE-A3 Inhibits p53 and Promotes Proliferation and Survival in Multiple Myeloma.

Abstract 1795

Poster Board I-821

The type I Melanoma Antigen GEne (MAGE) proteins MAGE-A3 and CT7 (MAGE-C1) were commonly detected in primary tumor cells from multiple myeloma patients and their expression was correlated with advanced disease and proliferation. They belong to the Cancer-Testis antigen (CTAg) family of tumor-associated proteins. In gene expression analyses of primary myeloma cells, CTAg were associated with proliferative gene signatures and poor clinical outcome. These findings suggest that type I MAGE may play a pathogenic role in proliferation or survival in multiple myeloma cells. To test this hypothesis, we examined MAGE expression, proliferation, and apoptosis in primary myeloma specimens and human myeloma cell lines (HMCL). First, we examined CTAg expression and proliferation in vivo at two critical clinical milestones, in newly diagnosed, untreated patients and patients who relapsed after chemotherapy. MAGE-A3 was detected in a higher percentage of tumor specimens from relapsed patients (77%) compared to those from newly diagnosed patients (36%, p=0.0003), whereas CT7 was detected in about 75% of both patient populations. The percentage of proliferating myeloma cells, as measured by staining for the proliferation marker Ki-67, was significantly higher in relapsed specimens (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p=0.0002), demonstrating an association between MAGE-A3, progression of disease and proliferation. Second, we investigated the functional role of MAGE-A3 by silencing this gene in HMCL by shRNA interference. Targeted lentiviral shRNA transduction efficiently knocked down MAGE-A3 mRNA (≥90% compared to controls) and protein in MM.1r and Arp-1 HMCL by 48 hours and this effect was maintained up to 96 hours. Pulse labeling of HMCL with bromodeoxyuridine for 30 minutes revealed that silencing of MAGE-A3 led to cell cycle arrest, as evidenced by the complete loss of cells in S phase and accumulation of cells in both G1 and G2. This was accompanied by increased expression of the tumor suppressor p53 and the endogenous cyclin-dependent kinase (CDK) inhibitor p21Cip1, a p53 target that inhibits CDKs in both late G1 and G2. However, CDK4/6-specific phosphorylation of the retinoblastoma gene product (Rb) was unimpaired, indicating that control of the mid-G1 cell cycle checkpoints by Rb remained intact and suggesting that MAGE-A3 acted in part to promote G1-S progression. Within 24 hours of cell cycle arrest, 70-80% of MAGE-A3-silenced cells underwent apoptosis as measured by Annexin V staining, compared to '20% in cells transduced with a non-target control lentivirus or untreated. Furthermore, this apoptosis was caspase-dependent, as it was completely prevented by the pan-caspase inhibitor Quinoline-Val-Asp-CH2-OPh, and was triggered by the loss of mitochondrial outer membrane potential in the activation of the intrinsic apoptosis pathway. Taken together, the in vivo and in vitro results suggest that MAGE-A3 promoted myeloma cell proliferation by inhibiting p53-dependent expression of p21, and loss of this activity leads to growth arrest and cell cycle-coupled apoptosis via activation of the intrinsic apoptosis pathway. Understanding the biochemical mechanism of MAGE-A3 in cell cycle regulation and survival may identify novel therapeutic strategies for multiple myeloma. Proof of principle in this disease may lead to broader application of these strategies in other cancers that express MAGE-A3.